1. Field of the Art
The present invention relates to DNA sequences which are useful for the synthesis of carotenoids such as lycopene, .beta.-carotene, zeaxanthin or zeaxanthin-diglucoside.
The present invention also relates to processes for producing such carotenoid compounds.
2. Related Art
Carotenoids are distributed widely in green plants. They are yellow-orange-red lipids which are also present in some mold, yeast and so forth, and have recently received increased attention as natural coloring materials for foods. Among these carotenoids, .beta.-carotene is a typical one, which is used as a coloring materials and as a precursor of vitamin A in mammals as well. It is also examined for its use as a component for preventing cancer [see, for example, SHOKUHIN TO KAIHATSU (Foods and Development), 24, 61-65 (1989)]. Carotenoids such as .beta.-carotene are widely distributed in green plants, so that the plant tissue culture has been examined for the development of a method for producing carotenoids in a large amount which is free from the influence of natural environment [see, for example, Plant Cell Physiol., 12, 525-531 (1971)]. The examination has been also made for detecting a microorganism such as mold, yeast or green algae which is originally high carotenoid productive and for producing carotenoids in a large amount with use of such microorganism (see, for example, The Abstract of Reports in the Annual Meeting of NIPPON HAKKO KOGAKU-KAI of 1988, page 139). However, neither of these methods are successful at present in producing .beta.-carotene at a good productivity which exceeds the synthetic method in commercial production of .beta.-carotene. It would be very useful to obtain a gene group which participates in the biosynthesis of carotenoids, because it will be possible to produce carotenoids in a large amount by introducing a gene group which has been reconstructed to express proper genes in the gene group in a large amount, into an appropriate host such as a plant tissue culture cell, a mold, an yeast or the like which originally produces carotenoids. Such a development in technology has possibilities for finding a method of producing .beta.-carotene superior to the synthetic method and a method of producing useful carotenoids other than .beta.-carotene in a large amount.
Furthermore, the synthesis of carotenoids in a cell or an organ which produces no carotenoid will be possible by obtaining the gene group participating in the biosynthesis of carotenoids, which will add new values to organisms. For example, several reports have recently been made with reference to creating flower colors which cannot be found in nature by using genetic manipulation in flowering plants [see, for example, Nature, 330, 677-678 (1987)]. The color of flowers is developed by pigments such as anthocyanine or carotenoids. Anthocyanine is responsible for flower colors in the spectrum of red-violet-blue, and carotenoids are responsible for flower colors in the spectrum of yellow-orange-red. The gene of the enzyme for synthesizing anthocyanine has been elucidated, and the aforementioned reports for creating a new flower color are those referring to anthocyanine. On the other hand, there are many flowering plants having no bright yellow flower due to no function of synthesizing carotenoids in petal (e.g. petunia, saintpaulia (african violet), cyclamen, Primula malacoides, etc.). If suitable genes having been reconstructed so as to be expressed in petal in a gene group referring to the biosynthesis of carotenoids are introduced into these flowering plants, the flowering plants having yellow flowers will be created successfully.
However, enzymes for synthesizing carotenoids or genes coding for them have been scarcely elucidated at present. The nucleotide sequence of the gene group participating in the biosynthesis of a kind of carotenoids has been elucidated lately only in a photosynthetic bacterium Rhodobacter capsulatus [Mol. Gen. Genet., 216, 254-268 (1989)]. But this bacterium synthesizes the acyclic xanthophyll spheroidene via neurosporene without cyclization and thus cannot synthesize general carotenoids such as lycopene, .beta.-carotene and zeaxanthin.
There are prior arts with reference to yellow pigments or carotenoids of Erwinia species disclosed in J. Bacteriol., 168, 607-612 (1986), J. Bacteriol., 170, 4675-4680 (1988) and J. Gen. Microbiol., 130, 1623-1631 (1984). The first one of these references discloses the cloning of a gene cluster coding for yellow pigment synthesis from Erwinia herbicola Eho 10 ATCC 39368 as a 12.4 kilobase pair (kb) fragment. In this connection, there is no illustration of the nucleotide sequence of the 12.4 kb fragment. The second literature discloses the yellow pigment synthesized by the cloned gene cluster, which is indicated to belong to carotenoids by the analysis of its UV-visible spectrum. The last literature indicates that the gene participating in the production of a yellow pigment is present in a 260 kb large plasmid contained in Erwinia uredovora 20D3 ATTC 19321 from the observation that the yellow pigment is not produced on curing the large plasmid, and further discloses that the pigment belongs to carotenoids from the analysis of its UV-visible spectrum.
However, the chemical structures of carotenoids produced by the Erwinia species or of its metabolic intermediates, enzymes participating in the synthesis of them or the nucleotide sequence of the genes encoding these enzymes remain unknown at present.